Use of chymotrypsin for the inactivation of prekallikrein activator

ABSTRACT

In order to avoid the risk of undesired side reactions of blood products, the latter are produced from plasma by using chymotrypsin to inactivate prekallikrein activator. These preparations are obtained by the fractionated enrichment of plasma proteins, with the proviso that a chymotrypsin solution or immobilized chymotrypsin is added to the fractions at any stage of the fractionation process. Before completion of the preparations, the chymotrypsin or the immobilized trypsin is removed from the preparations.

The invention relates to the production of blood products from human oranimal plasma, in particular of albumin and immunoglobulin-Gpreparations, which preparations are obtained by the fractionatedenrichment of plasma proteins.

Plasma contains Hageman factor (Factor XII), a proenzyme ofprekallikrein activator (PKA). Activated Hageman factors (Factor XIIaand Factor XIIf) act upon the blood coagulation scheme, the fibrinolyticand the kallikrein-kinin systems.

These prekallikrein activators may be formed at the production of bloodproducts from plasma, the preparations obtained, thus, having a PKAcontent. Blood products of this type, such as albumin andimmunoglobulin-G preparations, if infused in relatively large amounts,may cause adverse side reactions, e.g., states of shock (cf. B. M.Alving et al., The New England Journal of Medicine, vol. 299 (1978),66).

A method of inactivating incompatibility-reaction-causing substances inblood products, by using immobilized chymotrypsin is known from EP-A-0120 835. However, in the first place, vasoactive side effects andanticomplementary activity were considered as incompatibiliatyreactions.

It is, furthermore, known to inactivate PKA. Thus, in U.S. Pat. No.4,251,510 (Tankersley), the removal of PKA through adsorption by meansof silica-containing substances is described. This involves the dangerof getting traces of heavy metals getting into the preparations.Furthermore, the addition of inhibitors, such as C₁ -esterase inhibitor,is recommended in U.S. Pat. No. 4,608,254 (Philapitsch et al.). Suchinhibitors are hardly accessible.

The invention aims at avoiding such difficulties and has as its objectto enable the preparation of blood products that avoid the risk ofundesired side reactions and is based on the finding that chymotrypsinis apt to enzymatically cleave undesired prekallikrein activator and torender ineffective its capability of converting prekallilkrein intokallikrein.

Accordingly, the invention consists in the use of chymotrypsin for theinactivation of prekallikrein activator at the production of bloodproducts from human or animal plasma, in particular of albumin andimmunoglobulin-G preparations, which preparations are obtained by thefractionated enrichment of plasma proteins, with the proviso that achymotrypsin solution or immobilized chymotrypsin is added to thefractions at any stage of the fractionation process and the chymotrypsinor the immobilized chymotrypsin is removed from the preparations beforecompletion of the same.

According to a preferred embodiment, the invention consists in the useof chymotrypsin at the production of albumin and immmunoglobulin-Gpreparations, with the proviso that chymotrypsin in the form of anaqueous solution is added to an aqueous solution of the enriched plasmaproteins, the plasma proteins are precipitated by a proteinprecipitating agent and, thereby, are separated from the chymotrypsinsolution, whereupon the plasma proteins are processed to finishedpreparations.

According to another preferred embodiment, the invention consists in theuse of chymotrypsin at the production of albumin and immunoglobulin-Gpreparations, with the proviso that chymotrypsin immobilized atSepharose is added to an aqueous solution of the enriched plasmaproteins, the solid substances are then removed from the solution toseparate the chymotrypsin and the solutions containing the proteins areprocessed to finished preparations.

The invention will be explained in more detail by way of the followingexamples:

EXAMPLE 1 Production of an albumin preparation

To 7 l of human blood plasma 8% ethyl alcohol is added at a pH of 7.2and a temperature of -2° C., with a precipitate forming. Afterseparation of the precipitate, the ethyl alcohol concentration is raisedto 25% and the temperature is lowered to -6° C. The precipitate forming,which contains immunoglobulin, is separated, and the ethanolconcentration of the supernatant is increased to 40% at a pH of 6.5 anda temperature of -7° C. The formed precipitate, which contains alpha-and beta-globulin, is separated and discarded.

The pH of the supernatant is adjusted to 5.4, with albuminprecipitating. The latter is separated by centrifugation and issubjected to a further step of purification: the precipitate isdissolved in water and the ethanol concentration is adjusted to 10% at apH of 4.8 and a temperature of -2° C.

The precipitate is separated and discarded; the ethyl alcoholconcentration of the supernatant is raised to 40%, the temperature islowered to -8° C. and the pH is adjusted to 5.1. The albumin precipitateis separated by centrifugation. To remove the ethanol, the dissolvedprecipitate is subjected to ultrafiltration and the albumin solution issterile-filtered. The sterile albumin solution is incubated with 0.1unit of chymotrypsin (Sigma, product number: C-4129, lot 85 F-8130) per100 mg of albumin at +37° C. under stirring: after 48 hours theinactivation of PKA has been completed.

To remove the dissolved chymotrypsin, the albumin can be precipitated bymeans of 45% EtOH at -6° C. and separated from the supernatantcontaining the chymotrypsin. The albumin solution is then processed toan administrable preparation.

In the following, the effect of the addition of chymotrypsin accordingto the invention will be explained, wherein the technique and reagentsindicated below are applied for determining the prekallikrein activator:

Technique: From a purified prekallikrein preparation (PKK), kallikrein(KK) is generated by means of a prekallikrein activator (PKA). Thekallikrein amidolytically splits p-nitroanilide (pNA) from a specificchromogenic substrate. The concentration of pNA is measuredphotometrically at a wave length of 405 nm.

Reagents:

Buffer I: 6.06 g TRIS and 23.38 g NaCl are dissolved in about 500 ml H₂O dist. and are adjusted to a pH of 8.0 with diluted HCl and filled upto 1000 ml with H₂ O dist.

Buffer II: 1.81 g TRIS, 1.02 g imidazole and 6.43 g NaCl are dissolvedin about 500 ml H₂ O dist. and are adjusted to a pH of 7.9 with dilutedHCl and filled up to 1000 ml with H₂ O.

Chromogenic substrate:

S 2302 (Kabi)H-D-prolyl-L-phenylanyl-L-arginin-p-nitroanilid-dihydrochloride. A 10 mmolar aqueous solution is prepared.

25 mg S 2302 in 4.1 ml H₂ O dist.

Prekallikrein preparation: The production of the preparation is carriedout acording to a prescription of Harpel modified by M. S. Horowitz (NewYork Blood Center). Human citrated plasma is treated with the help ofDEAE cellulose. The fraction that has not been bound to DEAE cellulosecontains the prekallikrein.

Positive control (standard): As standard (=reference value), an albuminpreparation of the Bureau of Biologics (BoB) of the Food and DrugAdministration, Bethesda, Md. 20205, U.S.A., is used. This preparationcontains prekallikrein activator. The generation of kallikrein by thisBoB standard constitutes the reference value 1 and is equated to 100%.

Sample: If necessary, the sample is used in the assay in a dissolved ordilute state.

Assay: In a water bath at a temperature of 37° C.

100 myl prekallikrein preparation

50 myl buffer I

25 myl sample

are pipetted into a plastic tube. After an incubation time of 15 min at37° C.

300 myl buffer II

50 myl S 2302 substrate

are pipetted. This mixture is introduced into a photometer brought to atemperature of 37° C., and the increase in the optical density perminute Δ (OD/min) at a wave length of 405 nm is measured with a layerthickness of 10 mm.

The activity of a sample Δ (OD/min) is expressed factorially--relativeto the OBRR standard by the number 1--or in % of the OBRR standard; 1%of the OBRR standard equals 1 International Unit.

In the instant Example 1, PKA inactivation proceeded as follows:

    ______________________________________                                                PKA    Content of albumin (monomers)                                  ______________________________________                                        Beginning 43 IU/ml 87.6%                                                      after 24 h                                                                               4 IU/ml 87.4%                                                      after 48 h                                                                               1 IU/ml 87.0%                                                      ______________________________________                                    

EXAMPLE 2 Production of an albumin preparation

The preparation of the albumin solution was carried out as in Example 1;instead of the dissolved chymotrypsin, 0.25 ml of chymotrypsinimmobilized on Sepharose was used per 100 mg of albumin. Aftertermination of the PKA inactivation process, the water-insolublechymotrypsin was removed by filtration and the albumin was processes toan administrable preparation.

The immobilized chymotrypsin was prepared in the following manner: 100ml of Sepharose 4 B gel (Pharmacia), after washing with 400 ml distilledwater, were mixed with 20 g bromocyan dissolved in 10 ml acetonitrile ata pH of 11.0. The reaction mixture was cooled by an ice bath. Afterremoval of the liquid phase, the gel was mixed with 80 mg chymotrypsin(Sigma) dissolved in 100 ml 0.2 molar NaHCO₃. The non-bound trypsin isseparated by filtration from the trypsin bound to the gel.

After mixture of the gel trypsin with 100 ml of a 1 molar glycinesolution, it is thoroughly washed free of proteins with a 0.2 molarNaHCO₃ solution. Finally, the gel trypsin is suspended in 100 ml of a0.9% NaCl solution--it is ready for use in PKA inactivation.

PKA inactivation proceeded as follows, the determination of theprekallikrein activator having been carried out as in Example 1.

    ______________________________________                                                PKA    Content of albumin (monomers)                                  ______________________________________                                        Beginning 35 IU/ml 87.5%                                                      after 24 h                                                                               2 IU/ml 86.3%                                                      after 48 h                                                                               1 IU/ml 84.8%                                                      ______________________________________                                    

EXAMPLE 3 Production of an immunoglobulin-G preparation

Human blood plasma is mixed with 8% ethanol at a pH of 7.2 and atemperature of -2° C. After separation of the precipitate, the ethanolconcentration is increased to 25%, the temperature simultaneously beinglowered to -6° C. The precipitate, which contains immunoglobulin, isfurther purified by extraction with a phosphate-acetate buffer and ismixed with 12% ethanol at a pH of 5.3 and a temperature of -2° C.

The precipitate is discarded. The ethanol concentration of thesupernatant is increased to 25% at a pH of 7.2 and a temperature of -10°C. The pasty immunoglobulin precipitated is collected and dissolved; theethanol is removed by ultrafiltration.

For PKA inactivation, 1U of chymotrypsin (Sigma, product number C-4129)is added to the sterile solution per 100 mg immunoglobulin and istreated at +37° C. under stirring. After treatment, immunoglobulin wasprecipitated with 40% ethanol at -6° C. and thereby separated fromchymotrypsin. Immunoglobulin was processed to an administrablepreparation.

PKA inactivation proceeded as follows:

    ______________________________________                                                 PKA     Content of monomers IgG                                      ______________________________________                                        Beginning  323 IU/ml 88.5%                                                    after 24 h  4 IU/ml  87.0%                                                    after 48 h  0 IU/ml  84.6%                                                    ______________________________________                                    

EXAMPLE 4 Production of an immunoglobulin-G preparation

The production of the immunoglobulin-G-containing fraction was performedin the same manner as in Example 3.

Incubation was effected with immobilized chymotrypsin. 1.0 ml ofimmobilized chymotrypsin was added to 1,000 mg protein.

PKA inactivation proceeded as follows:

    ______________________________________                                                 PKA     Content of monomers IgG                                      ______________________________________                                        Beginning  315 IU/ml 88.6%                                                    after 24 h  22 IU/ml 87.9%                                                    after 48 h  0 IU/ml  85.8%                                                    ______________________________________                                    

What I claim is:
 1. A method for the inactivation of prekallikreinactivator during the production of immunoglobulin-G blood productpreparations from human or animal plasma comprising the steps of (1)obtaining said preparations in a fractionation process by the enrichmentin fractions of plasma proteins, (2) adding dissolved chymotrypsin orimmobilized chymotrypsin to said fractions at any stage of saidfractionation process and (3) removing the respective one of saiddissolved chymotrypsin and immobilized chymotrypsin from saidpreparations before completion thereof.
 2. A method for the inactivationof prekallikrein activator during the production of immunoglobulin-Gblood product preparations from human or animal plasma comprising thesteps of (1) obtaining said preparations in a fractionation process bythe enrichment in fractions of plasma proteins, (2) adding an aqueouschymotrypsin solution to an aqueous solution of enriched plasma proteinsat any stage of said fractionation process, (3) precipitating saidplasma proteins by a protein precipitating agent so as to be separatedfrom said chymotrypsin solution, and (4) processing said plasma proteinsto finished preparations.
 3. A method for the inactivation ofprekallikrein activator during the production of immunoglobulin-G bloodproduct preparations comprising the steps of (1) obtaining saidpreparations in a fractionation process by the enrichment in fractionsof plasma proteins, (2) adding chymotrypsin immobilized on Sepharose toan aqueous solution of enriched plasma proteins at any stage of saidfractionation process, (3) removing solid substances from said solutionto separate said chymotrypsin and to obtain protein-containingsolutions, and (4) processing said protein-containing solutions tofinished preparations.